P<0.01; 17.1±19.6 vs 27.6±21.3 sec, P<0.01, respectively). Univariate logistic regression found that the odds ratio (OR) for the slower walking speed (<1 m/sec) in the VI individuals was significantly higher at 7.40  than in non-VI individuals. It was still significantly higher at 4.50  in the multivariate logistic regression model after adjusting for the BCVA, age, sex, current smoking habit, and health conditions. Our results indicate that the walking speed and one-leg standing times were significantly associated with VI.The aim of this study was to determine whether there is a significant association between a visual impairment (VI) and mobility functions in an elderly Japanese cohort. The subjects of this study were part of the Fujiwara-kyo Eye Study, a cross sectional epidemiological study of elderly individuals conducted by Nara Medical University. Participants were ≥70-years who lived in the Nara Prefecture. All underwent comprehensive ophthalmological examinations, and a VI was defined as a best-corrected visual acuity (BCVA) worse than 20/40 in the better eye. The associations between the BCVA and walking speed and one-leg standing time were determined. The medical history and health conditions were evaluated by a self-administered questionnaire. A total of the 2,809 subjects whose mean age was 76.3 ± 4.8 years (± standard deviation) were studied. The individuals with a VI (2.1%) had significantly slower walking speeds and shorter one-leg standing times than that of the non-VI individuals (1.5±0.4 vs 1.7±0.4 m/sec,  It has been reported that the risk of the risk of mobility difficulties increase after 65-years-of-age , and mobIn earlier studies, we found that VIs affected the cognitive function of the elderly subjects ,6. Our nThe Salisbury Eye Evaluation Studies reported that individuals with VIs had greater mobility disabilities than the non-VIs individuals ,13. HoweThe walking speed is a strong predictor of disability and death in older adults –17, and The purpose of this study was to determine the effect of VIs on the physical capabilities. To accomplish this, we measured the walking speed and the one-leg balancing times in an elderly population whose VIs were also measured.A detailed description of the Fujiwara-kyo Study has been published ,25. BrieAn ophthalmological survey, the Fujiwara-kyo Eye study, was conducted for the first time as part of the Fujiwara-kyo Study at the second survey during February to November 2012. The data presented in this manuscript were collected from the examinations done in 2012 ,6. The sThe surveys were conducted in accordance with the tenets of the Declaration of Helsinki, and the protocol was approved by the Ethics Review Board of Nara Medical University. A signed informed consent form was obtained from all participants.Visual acuity. The refractive error  was determined by an auto refractometer and keratometer . The data of the refractive errors were used as useful references to obtain the best-corrected visual acuity (BCVA). The uncorrected and the BCVA of both eyes were measured with a Landolt ring chart at 5 m by well-trained orthoptists. To obtain the BCVA, orthoptists used corrective lenses placed in trial frames for each participant. A VI was defined as BCVA worse than 20/40 in the better-seeing eye for the statistical analyses. This cut-off BCVA corresponded to the American Association of Ophthalmology categorization of a visual impairment which was defined as the distance BCVA worse than 20/40 in the better-seeing eye [eing eye . The decThe walking speed was assessed over a distance of 10 m after 2 m for acceleration. Participants were required to walk as fast as possible at their maximal speed. Participants walked on a flat floor wearing walking shoes or sneakers. An attendant followed each participant during the speed walking test and one-leg standing test to protect the participants from an unexpected occurrences and avoid injuries from falls. In all tests, no accidental falls happened, and all participants finished the mobility tests safely. The walking time in 0.1 sec periods was measured with an automatic measurement system , and the walking speed (m/s) was calculated by dividing distance (10 m) by the time. The mean results of two trials was used for the statistical analysis. We defined a slow walking speed as <1 m/sec according to the sarcopenia criteria of Japanese version of the CHS (J-CHS criteria) , the IntParticipants were instructed to stand on their preferred leg for as long as possible with their eyes opened and hands on their waist. The duration of the one-leg stand was until balance was lost. The one-leg standing time represents the static balance of the body, and a shorter one-leg standing time suggests an increased risk of falling ,23. If tThe medical history and medication information were obtained by a self-administered questionnaire. The presence of systemic hypertension was based on a self-reported diagnosis and current antihypertensive therapy. The presence of diabetes mellitus was based on a self-reported diagnosis, current anti-diabetic therapy, and fasting plasma glucose and glycated hemoglobin levels. The presence of hip arthritis, knee arthritis, leg pain, ankle and foot pain, low back and buttock pain, and sciatica were defined base on self-administered questionnaire and self-reported previous diagnosis.2), normal (18.5 to <25 kg/m2), and high (≥25 kg/m2).We also determined the presence of covariates; body mass index (BMI), smoking status, and a number of comorbid conditions. BMI was calculated as body weight (kg) divided by height (m) squared, and it was categorized into three groups: low  were studied. There were 1,482 men and 1327 women, and 60 (2.1%) of the participants were categorized as VI, and 2,749 (97.9%) were categorized as non-VI. The VI participants were significantly older but there was no significant difference in the sex distribution, BMI, smoking habit, and general health conditions between the two groups .P <0.05; P <0.05; The participants in the VI group had significantly slower walking speeds of 1.5 ± 0.4 m/sec than did the non-VI group at 1.7 ± 0.4 m/sec P <0.05; . In addiP <0.001) as determined by linear regression analyses of univariate models. In the multivariate analyses, the visual acuity, age, sex, and the number of other health conditions were significantly associated with the walking speed and one-leg standing time  than for the non-VI group  than for non-VI group Click here for additional data file.


We also analyzed the p.D386N and p.E420K of SPINK5 variants and loss-of-function mutations of FLG in the AD patients. The serine protease activity in the SC was increased not only in AD lesions but also in non-lesions of AD patients. We found, generally, that there was a positive correlation between the serine protease activity in the SC and the total serum immunoglobulin E (IgE) levels, serum thymus and activation-regulated chemokine (TARC) levels, and peripheral blood eosinophil counts. Moreover, the p.D386N or p.E420K in SPINK5 and FLG mutations were not significantly associated with the SC’s serine protease activity. Epidermal serine protease activity was increased even in non-lesions of AD patients. Such activity was found to correlate with a number of biomarkers of AD. Further investigations of serine proteases might provide new treatments and prophylaxis for AD.The serine proteases kallikrein-related peptidase (KLK) 5 and KLK7 cleave cell adhesion molecules in the epidermis. Aberrant epidermal serine protease activity is thought to play an important role in the pathogenesis of atopic dermatitis (AD). We collected the stratum corneum (SC) from healthy individuals ( KLK1-15) in the human genome [KLK5 mice display cutaneous and systemic hallmarks of severe inflammation and allergy with pruritus [KLK7 in epidermal keratinocytes have been found to develop pathologic skin changes with increased epidermal thickness, hyperkeratosis, dermal inflammation, and severe pruritus [Atopic dermatitis (AD) is a chronic, pruritic inflammatory skin disease that affects up to 25% of children and 2–3% of adults . AD has n genome . KLK5, an genome . It has n genome ,11. Resen genome . Moreovepruritus . Transgepruritus . Our groserine protease inhibitor Kazal-type 5 (SPINK5) gene, is composed of 15 Kazal-type domains, all of which are capable of inhibiting serine protease activity including KLK5 and KLK7 [SPINK5 develop Netherton syndrome, characterized by ichthyosis, hair abnormality, and atopic manifestations [SPINK5 such as the p.D386N  and p.E420K  have been reported to be associated with the pathogenesis of AD [Serine protease activity in the skin is tightly regulated by not only KLKs but also serine protease inhibitors such as lympho-epithelial Kazal-type-related inhibitor (LEKTI), secretory leukocyte protease inhibitor (SLPI), and elafin . LEKTI, and KLK7 ,18. Indistations . Nonsynois of AD ,23,24,25FLG) were identified as the cause of ichthyosis vulgaris [FLG are predisposing factors in the context of AD [FLG mutations [FLG mutations have been identified in the Japanese population [FLG mutations are well known to affect epidermal barrier functions, the association between the mutations and the epidermal serine protease activity has not been investigated.Profilaggrin is dephosphorylated and degraded to produce monomeric filaggrin in the SC and then further proteolyzed to release its component amino acids . Profilavulgaris . The samxt of AD . In 2009utations . To datepulation . AlthougHere, we performed multifaceted analyses of epidermal serine protease activity in patients with AD. We focused particularly on the relationship between the epidermal serine protease activity and biomarkers of AD or AD-related gene variants.We examined trypsin- and chymotrypsin-like serine protease activity in SC samples from normal healthy volunteers and AD patients. The activity of trypsin- and chymotrypsin-like serine proteases in the SC samples from both non-lesions and lesions of the AD patients were significantly higher than those from the healthy individuals A,B. The n = 63) and various medical treatments. The treatments were as follows: topical corticosteroid (n = 44), topical tacrolimus (n = 15), oral antihistamine (n = 36), oral corticosteroid (n = 6) and oral cyclosporine (n = 4). The trypsin-like serine protease activity in the SC of patients with topical corticosteroid therapy was significantly higher than those without topical corticosteroid therapy  levels were highly correlated with the trypsin-like serine protease activity in non-lesions of the AD patients, but not with the chymotrypsin-like serine protease activity in non-lesions or the trypsin- or chymotrypsin-like serine protease activity in AD lesions A–D. The FLG mutations in the AD patients. Eighteen of the 115 patients (15.7%) had one of the ten FLG mutations. We were able to perform protease assays with the SC samples from 61 of the AD patients. Ten of the 61 patients (16.4%) had the FLG mutations: p.Q1701X (n = 1), p.S2554X (n = 1), p.S2889X (n = 4), p.S3296X (n = 2) and p.K4022X (n = 2). We also analyzed p.D386N (c.G1156A) and p.E420K (c.G1258A) of SPINK5 in the AD patients. In respect of p.D386N, the numbers of the genotypes were as follows: GG (n = 21), GA (n = 28), and AA (n = 12). For p.E420K, the numbers of the genotypes were as follows: GG (n = 14), GA (n = 31), and AA (n = 16). The trypsin- and chymotrypsin-like serine protease activity in the SC was not significantly different among the subgroups of p.D386N -4 and IL-13 increase the expression and function of KLK7 in epidermal keratinocytes , the uprWe next investigated the association between serine protease activity in the SC and the content of therapy for AD. The trypsin-like serine protease activity in the SC of patients with topical corticosteroid therapy was significantly higher than those without topical corticosteroid therapy. These results suggest the possibility that patients with clinically severe AD needed to be treated with topical corticosteroid and clinically mild patients were able to be treated with topical tacrolimus or moisturizer only. Thus far, it is unknown why there is a difference between trypsin-like and chymotrypsin-like serine protease activity even though they are positively correlated see . FurtherWe also investigated the correlation between serine protease activity in the SC and several biomarkers of AD. Not all serine protease activity was significantly correlated with the serum total IgE levels, serum TARC levels, or peripheral blood eosinophil counts, but there were tendencies for these to correlate positively. The absence of statistical significance in several of the comparisons might be due to the insufficiency of the sample number considered for the analysis or the technical instability of our protease assays. Serum total IgE reflects the degree of allergen sensitization ,32. SeruSPINK5 and loss-of-function mutations in FLG are involved in serine protease activity in the SC. p.D386N and p.E420K polymorphisms of SPINK5 were each reported to be associated with the pathogenesis of AD [We investigated whether nonsynonymous variants of is of AD ,23,24,25FLG mutations are well known to be strongly associated with the pathogenesis of AD [FLG mutations. Therefore, one or more other genetic factors can be expected to be associated with the pathogenesis. We had hypothesized that AD patients without FLG mutations have serine proteases-related gene variants such as SPINK5 and aberrant protease activity, but our analyses revealed that there is no significant association between these mutations and the serine protease activity in the SC. On the contrary, there was a tendency for the AD patients with FLG mutations to have higher serine protease activity compared to the patients without FLG mutations. Further analyses might clarify the association between the increase in serine protease activity and genetic factors in AD patients.The is of AD ,29, but SPINK5 and FLG mutations did not affect the serine protease activity of the SC in Japanese individuals with AD. Further investigations of epidermal serine protease activity might enable the design of new therapeutic and prophylactic drugs for the treatment of AD.In conclusion, the results of our present analyses demonstrated for the first time that epidermal serine protease activity is increased even in non-lesions of AD patients and this activity is associated with biomarkers of AD. In addition, p.D386N and p.E420K of The SC samples and blood samples were collected with written informed consent from normal healthy volunteers and patients with AD at Okayama University Hospital and its affiliated hospitals. This study was approved by the Ethics Committee of Okayama University .n = 46; ages 35.0 ± 8.8 years). The SC of patients who had been diagnosed with AD based on the diagnostic criteria of Hanifin and Rajka were obtained from the upper extremities (lesions and non-lesions) . Cellotape™  was used to collect the SC samples. The 10-cm-long tape was put on and peeled from the surface of the extremities (approx. 10–20 times) until the adhesion disappeared. The tape was then stored at −20 °C until the following treatment with hexane.Samples of the SC of healthy individuals were obtained from the forearms of normal healthy volunteers  and incubated at 37 °C for 1 h on a shaker and then used in the protease assays.For the measurement of protease activity in the SC samples, 100 μL of sample solution was mixed with 100 μL of a substrate that is specific for trypsin-like serine proteases  or chymotrypsin-like serine proteases  in 10 mmol/L Tris-HCl (pH 7.8). The final concentration of each substrate was 0.4 mmol/L. The mixture was incubated at 37 °C for 24 h and the fluorescence (excitation/emission = 380/460 nm) was then measured by a FlexStation 3 Multi-Mode Microplate Reader  at the Central Research Laboratory, Okayama University Medical School. For the correction of the differences in the SC sample amounts, the value of measured fluorescence (AU) was divided by dried weight of the SC sample (mg).The patients’ laboratory data were extracted from their medical records. The serum total IgE level, serum TARC level and peripheral blood eosinophil count that had been obtained at the time point closest to the patient’s tape stripping were selected. We also collected the medical treatment histories of the patients such as their use of topical corticosteroid and topical tacrolimus, oral antihistamine, oral corticosteroid and oral cyclosporine.SPINK5, the 453-bp DNA was amplified with two specific primers (SPINK5-E13-FW: 5′-TTCCTATCTCTTGGCATATGATGT-3′ and SPINK5-E13-RV: 5′-TGTCTCCAATCAGACAGTTTCTC-3′). For exon 14 of SPINK5, the 294-bp DNA was amplified with two specific primers (SPINK5-E14-FW: 5′-CAGGGTTAGGCACATCACATTC-3′ and SPINK5-E14-RV: 5′-TAAGGAATGCACGTGTTCCCTG-3′).Genomic DNA was extracted from whole blood using the Wizard Genomic DNA Purification Kit . For exon 13 of The polymerase chain reaction (PCR) products were purified using exonuclease I and shrimp alkaline phosphatase  and sequenced using a BigDye Terminator v3.1 Cycle Sequencing Kit  and an ABI3100 DNA sequencer (Applied Biosystems) at the Central Research Laboratory, Okayama University Medical School.FLG mutations was performed. In the present FLG mutation screening, we investigated the 10 FLG mutations as described [FLG mutations was performed with a TaqMan probe genotyping assay. Real-time PCR-based genotyping of the FLG mutations was performed with a TaqMan probe genotyping assay according to the manufacturer’s instructions . To detect an allele of each mutation, a set of two TaqMan probes labeled with a fluorescent dye (FAM or CAL Fluor Orange 560) and a quencher dye (BHQ-1), in addition to sequence-specific forward and reverse primers, were synthesized by Biosearch Technologies . The sequence of assay probes/primers was as provided [Real-time PCR-based genotyping of escribed . Brieflyprovided . The reat-test was used to determine the significance of differences between pairs of groups. The correlation between serine protease activity and the blood examination results was analyzed by Spearman’s rank correlation coefficient. Values of p < 0.05 were considered significant.All statistical analyses were conducted with GraphPad Prism 4 ver. 4.03 software . Student’s


E. coli, killed by treatment with a propanol-based hand rub, on antimicrobial resistance in E. coli isolates.To determine the effect of exposure to remnants of a phagemid-containing E. coli isolate (EUR1) was exposed to remnants of an E. coli K-12 strain containing a phagemid (pBS-E12) strain treated with Sterillium®. A series of 200 experiments was performed using this in vitro model. As a control, a series of 400 experiments was performed where the EUR1 was exposed either to the remnants of an E. coli K-12 strain (not containing a phagemid) (E12) treated with Sterillium® (n = 200) or to dried Sterillium® only (n = 200). The number of experiments that showed growth of an amoxicillin-resistant EUR1 isolate was evaluated in all three groups. An additional 48 experiments were performed in which a different clinical E. coli isolate (EUR2) was exposed to remnants of the pBS-E12 treated with Sterillium®. Whole-genome sequencing and phenotypic testing for AmpC beta-lactamase production was performed to investigate the mechanism behind this resistance development.An in vitro model was developed in which a clinical In 22 (11.0%) of 200 experiments in which the EUR1 isolate was exposed to remnants of a pBS-E12 an amoxicillin-resistant mutant isolate was obtained, as opposed to only 2 (1.0%) of 200 experiments involving the exposure of the EUR1 to Sterillium® only ) and 1 (0.5%) of 200 experiments involving the exposure of the EUR1 isolate to the remnants of the phagemid-free E12 . In 1 (2.1%) of the 48 experiments in which the EUR2 isolate was exposed to remnants of a pBS-E12 an amoxicillin-resistant mutant isolate was obtained. The development of resistance in all experiments was due to mutations in the promoter/attenuator region of the chromosomal AmpC beta-lactamase (cAmpC) gene leading to cAmpC hyperproduction.E. coli isolate to another phagemid-containing E. coli that was treated with propanol-based hand rub increased the development of amoxicillin resistance. Although phagemids are cloning vectors that are not present in clinical isolates, this finding may have implications for hand disinfection practices in healthcare facilities.Exposure of an  Escherichia coli . Bo. Bo30]. E. coli isolates after exposure to the remnants of the pBS-E12 treated with a propanol-based hand rub as compared to exposure to remnants of the E-12 treated with a propanol-based hand rub and as compared to exposure to only a propanol-based hand rub. AmpC beta-lactamase hyperproduction, due to mutations in the AmpC promoter/attenuator region, was responsible for the development of the observed AMR. The number of resistant mutants that developed in the isolates exposed to the remnants of the pBS-E12 also exceeded the number of pBS-E12 growing in the disinfection experiments, which indicates that amoxicillin degradation by the pBS-E12 beta-lactamase was not responsible for the observed increase in AMR. All mutated EUR1 isolates showed a high number of allele differences when compared to the wildtype isolate, suggesting genome-wide mutations. Interestingly this genome-wide mutational pattern was not detected in the EUR2 isolate. Indicating that the genome-wide mutational pattern might not only be (control) experiment but also isolate related.An in vitro model was developed to simulate propanol-based hand disinfection as is common practice in healthcare facilities nowadays. Bacteria are effectively killed, but their remnants remain present after disinfection. Subsequently susceptible bacteria are exposed to the remaining debris, including DNA. Experiments performed using this model showed a significant increase in the development of amoxicillin resistance in The exact underlying mechanism leading to the AMR conferring mutations in the promoter/attenuator region of the cAmpC remains unknown. Since the amoxicillin resistance gene of the pBluescript phagemid was not detected in the EUR1 and EUR2 mutant isolates, it seems more likely that the remnants of the pBS-E12 increased the mutation rate in the EUR1 and EUR2 isolate facilitating AMR development through cAmpC hyperproduction. Previous studies have implicated several mechanisms that may increase the mutation rate and result in AMR, such as the general stress response and an increased constitutive mutation frequency –35. FurtE. coli isolates and increased development of AMR has not been investigated to date. Interestingly, this increase in the development of AMR was only related to exposure to remnants of phagemid-containing E. coli isolates. Other studies have already shown that extracellular DNA can lead to the development of AMR in other bacteria [E. coli.Several studies have shown that environmental stressors other than antibiotics can influence the development of AMR –38. Howebacteria , 23. HowDespite the increased development of AMR was only related to exposure to remnants of the pBS-E12, it remains unknown if and what specific compounds of Sterillium® contribute to the observed increased development of AMR. Contrary to other alcohol-based hand rubs, Sterillium® contains mecetronium etilsulfate which potentially has a lasting antimicrobial effect , 40. FurE. coli isolates following exposure to stepwise increasing concentrations of amoxicillin over several days [E. coli isolate. Even in our control experiments, in which the E. coli isolates were only exposed to Sterillium®, cAmpC hyperproduction developed after only short sub-inhibitory amoxicillin concentrations. Moreover, in the isolates EUR1M2-EUR1M5 and EUR1M7–9 three mutations were detected in the cAmpC promotor region. Each of these mutations individually could increase cAmpC production in E. coli [Although cAmpC hyperproduction has been implicated in beta-lactam resistance in ral days , 42. cAm E. coli , 43. Per E. coli  exposureE. coli [E. coli isolates for amoxicillin resistance induction. Moreover, our in vitro model simulates propanol-based hand disinfection procedures which are very common in clinical practice [Contrary to other studies investigating the development of AMR in E. coli , 44, we practice , 45. Alspractice .E. coli isolates containing wildtype phagemids also increase the development of AMR needs further investigation. Moreover, since the pBluescript KS(−) phagemid contains both a bacteriophage origin of replication and a plasmid origin of replication future studies are required to assess whether the observed effect is bacteriophage or plasmid related. Also, only a limited number of isolates were used in this study. The extent to which this AMR induction is possible in other E. coli isolates or other species remains unknown. Moreover, it remains unknown whether and to what extent this in vitro phenomenon plays a role in the development of AMR in vivo.This study has some limitations. The pBluescript-KS (−) phagemid is a cloning vector not present in clinical isolates. Whether remnants of E. coli isolate after exposure to a phagemid-containing E. coli treated with a propanol-based hand rub.To the best of our knowledge, this is the first study showing development of amoxicillin resistance in an E. coli isolate after exposure to an unrelated phagemid-containing E. coli treated with a propanol-based hand rub. Although phagemids are cloning vectors that are not present in clinical isolates, this finding may have implications for hand disinfection practices in healthcare facilities.This exploratory study showed the development of amoxicillin resistance in an Additional file 1:Table S1. Characteristics of used strains. Table S2. Features of pBleuscript KS(-) phagmid ATCC® 87047TM. Table S3. Inhibition zone diameters of D68C AmpC & ESBL detection set for the pBS-E12, EUR1, EUR2 and mutant isolates.


The aim of this study was to assess different amyloid beta subspecies’ effects on behaviour and cognition in mice and their interaction with isoflurane anaesthesia.After governmental approval, cannulas were implanted in the lateral cerebral ventricle. After 14 days the mice were randomly intracerebroventricularly injected with Aβ 1–40 (Aβ40), Aβ 1–42 (Aβ42), 3NTyr10-Aβ (Aβ nitro), AβpE3-42 (Aβ pyro), or phosphate buffered saline. Four days after the injection, 30 mice  underwent general anaesthesia with isoflurane. A “sham” anaesthetic procedure was performed in another 30 mice . During the next eight consecutive days a blinded assessor evaluated behavioural and cognitive performance using the modified hole-board test. Following the testing we investigated 2 brains per subgroup for insoluble amyloid deposits using methoxy staining. We used western blotting in 4 brains per subgroup for analysis of tumour-necrosis factor alpha, caspase 3, glutamate receptors NR2B, and mGlu5. Data were analysed using general linear modelling and analysis of variance.Aβ pyro improved overall cognitive performance (p = 0.038). This cognitive improvement was reversed by isoflurane anaesthesia (p = 0.007), presumably mediated by decreased exploratory behaviour (p = 0.022 and p = 0.037). Injection of Aβ42 was associated with increased anxiety (p = 0.079). Explorative analysis on a limited number of brains did not reveal insoluble amyloid deposits or differences in the expression of tumour-necrosis factor alpha, NR2B, mGlu5, or caspase 3.Testing cognitive performance after intracerebroventricular injection of different amyloid beta subspecies revealed that Aβ pyro might be less harmful, which was reversed by isoflurane anaesthesia. There is minor evidence for Aβ42-mediated neurotoxicity. Preliminary molecular analysis of biomarkers did not clarify pathophysiological mechanisms. The accumulation of amyloid beta (Aβ) in the brain is one of the key factors in the pathophysiology of Alzheimer’s disease (AD) . Aβ is gAn increasing number of aged patients requires surgery and anaesthesia –22. ConsThe aim of this investigation was to further elucidate the effects of different Aβ subspecies on cognition in mice and their interaction with anaesthetics. Since there is no mouse model displaying pathology derived from post-translationally modified Aβ proteins, we decided to use the method of intracerebroventricular injection (ICV). Previous experiments have demonstrated that intracerebroventricular injection of Aβ oligomers leads to cognitive deficits , althougin vivo in C57/BL6 mice . It is a modification of Abeta 1-42. Total Abeta contains 10-15% of pyroglutamated amyloid beta (i.e. Abeta pyro) and it represents a dominant fraction of Aβ peptides in senile plaques of AD brains [2]. Abeta nitro (3NTyr10-Aβ) is also a nitrotyrosinated (or nitrated) form of amyloid beta 1-42 [3] and is found in the cores of amyloid plaques in AD brains [4]. Pyroglutamylation as well as nitrotyrosination of amyloid beta leads to increased oligomer stability and thus neurotoxicity in vitro as well as in vivo shown by neurodegeneration, premature mortality of mice, and disruption of calcium dyshomeostasis . Abeta1-40, Abeta1-42, Abeta-pyro and Abeta-nitro are all post-translationally modified forms of the peptide amyloid-beta. We described Abeta pyro and Abeta nitro in lines 54 – 58 (unmarked version of the revised manuscript)/57 – 61 (marked-up copy of the revised manuscript) more clearly and cited relevant literature.• “to ultimately find the best anaesthetic regimen for … AD”, this statement in the introduction should be clarified as only one anaesthetic agent is included in this study. Furthermore, an explanation of why isoflurane chosen rather than another aesthetic to study, would strengthen the introduction.We are grateful to Reviewer #1 for this extremely helpful remark. With this sentence we wanted to express our long-term goal of understanding the pathophysiology underlying Alzheimer’s disease and its interaction with different anaesthetics. Since the aim of our study was to investigate the effects of different intracerebroventricularly administered Abeta subspecies  in vivo and their interaction with general anaesthesia, we chose to concentrate on one anaesthetic agent, i. e. isoflurane. Isoflurane has been shown to induce caspase activation and increase levels of beta-site APP-cleaving enzyme (BACE) in vivo in C57/BL6 mice [7]. Sevoflurane seems to induce cellular and histological effects comparable to isoflurane [8], while desflurane was associated with a decrease in Abeta 1-42 levels [9]. As recommended, we included parts of the paragraph above in the introduction (lines 78–82 unmarked version/lines 92–96 marked-up version of the revised manuscript) and discussed desflurane as a possible anesthetic for future research (lines 404-406 unmarked version/lines 429-431 marked-up version of the revised manuscript).• What is the rationale for investigating the interaction between different Abeta species and anaesthetics? Please include relevant literature in the introduction to address this. Thank you for this important critique which helps us to improve our manuscript. Alzheimer’s disease (AD) is the most common form of dementia worldwide and affects as much as three percent of men and women aged between 65 and 74 years [10]. Due to an ongoing medical progress this population is also very likely to undergo surgery, often conducted under general anesthesia [10]. It is still unclear whether general anesthesia contributes to the development of AD. Some studies suggest a possible link between anesthesia and AD , while more recent ones do not . Also, it is nearly impossible to discriminate the influence of general anesthesia from the effect of surgery itself on the development of AD, as Lee et al. stated earlier this year [15]. To further illuminate the pathophysiology behind AD and the possible association of anesthesia and AD we wanted to investigate the potency of inducing AD and their individual interaction with isoflurane of the most prominent amyloid beta subspecies  one by one. We included relevant literature in the introduction in lines 62, 65, 66, 68 (unmarked copy of the revised manuscript)/67, 70 - 73 (marked-up version of the revised manuscript).• The rationale for why TNFalpha, caspase 3, NR2B and mGlu5 were chosen as biomarkers is explained in the Discussion, but it would benefit the reader if this information was presented earlier in the manuscript.We included our reasons for investigating TNFalpha, caspase 3, NR2B and mGlu5 in the introduction (lines 83 - 85 (unmarked version)/102 - 104 (marked-up version)). • The anaesthetics/AD literature is currently not adequately referenced in the Introduction , this should be rectified.Thank you for raising this important point. We corrected for this in the unmarked version of the revised manuscript in lines 52, 53, 59, 61, 62, 64, 317/in the marked-up copy of the revised manuscript in lines 55, 56, 63, 66, 67, 70, 340. We also reworded lines 314 - 317 in the unmarked copy of the revised manuscript/338 - 340 in the marked-up version of the revised manuscript. MethodsThe major concerns I have with this work is the experimental design; the study is likely underpowered, lacks some controls and uses different concentrations of the Abeta species across the experimental groups.• This study is underpowered, behavioural data has n=6 per experimental group, amyloid deposit analysis n=2 per experimental group and WB analysis n=4 per experimental group. An n=2 per experimental group is not sufficient for analysis. Power calculations should be performed to determine the change that can be detected with 90% power for each dataset in the manuscript and this information needs to be included in the manuscript. This will allow the authors to comment on whether the study was sufficiently powered to detect a change, or whether the results a likely to represent false negatives.We apologize for not describing the statistical approach in sufficient detail, especially the sample size considerations. Accordingly, the reviewer must have come to the impression of a power problem. We included our sample size considerations in the methods section (lines 245 - 249 in the unmarked version of the revised manuscript/ lines 268 - 272 in the marked-up version of the revised manuscript):The primary endpoint of the study was the cognitive and behavioural outcome. The variables of the hole-board test are considered relevant if two groups differ two times the given standard deviation. Based on a type I error of 0.05, a type II error of 0.20 and two-sided t-tests at the final test level of the hierarchical model 4 animals per group would have been appropriate. Our internal standard, however, suggests a minimal group size of six, which has been used accordingly.In order to facilitate readability of our findings in the modified hole-board test, we calculated the effect size of our findings and included mean differences with 95% confidence interval and partial eta-squared in the manuscript . We agree with the reviewer, that our study was not sufficiently powered for amyloid deposit and western blot analysis. We did explorative studies on a limited number of brains on amyloidopathy and other biomarkers in order to detect targets for future research concerning the pathomechanism of different Abeta subspecies and the interaction of isoflurane and amyloid-beta. Since we could not detect any amyloid deposits, we did not perform statistical analyses regarding amyloidopathy. Although we are aware of the shortcomings, we think that the results of the amyloid deposit and the western blot analysis are valid and should be presented in the manuscript. However, we stated the preliminary character of these analyses more clearly in the abstract and the discussion of the revised manuscript .• The Abeta 1-42 solution injected includes hexafluoroisopropanol and NaOH, whereas all other Abeta species are diluted in PBS. An additional control group with the same concentration of hexafluoroisopropanol and NaOH in PBS should be included in the study.We are deeply grateful to Reviewer #1 for raising this important point and apologize for providing insufficient detail in the original version of the manuscript. After suspending Abeta 1-42 in hexafluoroisopropanol (HFIP), it was removed from the stock solution using a vacuum concentrator . HFIP has been shown to increase cell permeability and to decrease cell viability , so a protocol to purge HFIP from the Abeta 1-42 stock solution was established in our lab [18]. We reworded the respective part in the Methods section in the revised manuscript . After Abeta 1-42 was dissolved in NaOH it was diluted 1:100 with PBS. Therefore, the amount of NaOH injected can be considered as minimal. We added the dilution ratio in the manuscript . As we did not shield the solution containing NaOH from air, NaOH reacted with CO2 in the 15 to 45 minutes before injection and formed sodium carbonate and sodium bicarbonate, which further reduced the amount of NaOH. In conclusion we consider the effect of the NaOH used to solve Abeta 1-42 minimal. To perform our experiments in accordance with the principles of the 3Rs  in animal research we decided against an additional control group. However, in order to account for a possible bias, we added the use of NaOH in only one group as a possible limitation in the discussion of the revised manuscript .• Different concentrations of each Abeta species are used, the explanation for this is that the different concentrations of Abeta species will have equivalent “neurotoxicity” and a paper examining LTP, EPSC and spine density data from brain slices is references. Please define “neurotoxicity” in this context. How do the authors know that this measure of “neurotoxicity” from brain slices will be the same in vivo?Thank you very much for drawing our attention to this important point. Our definition of “neurotoxicity” in this context was the ability of Abeta 1-40, Abeta 1-42, Abeta pyro and Abeta nitro to induce alterations in behaviour, cognition and fine motor skills in mice and the ability to induce amyloid deposits in the brains, respectively. With our study we wanted to investigate whether the findings of Rammes et al.  species on long-term potentiation (LTP)” Neuropharmacology. 2018 Sep 15;140:184-192. doi: 10.1016/j.neuropharm.2018.07.021. Epub 2018 Aug 11.) concerning the AD-inducing effects of different Abeta subspecies in vitro could be transferred into a mouse model. Therefore, we chose to use the same concentrations as Rammes et al. We did not know if the effects on brain slices were to be the same in vivo but we wanted to find out with this study. In order to avoid confusion for the reader we replaced the term “neurotoxicity” in line 152 - 153 (unmarked version of the revised manuscript)/line 175, 176 (marked-up copy of the revised manuscript) and replaced it with the actual in vitro findings of the cited reference. • Insufficient detail in the methods:o Where were the Abeta species purchased from or how were they made?We apologize for not stating this beforehand. Aβ1-40 and Aβ1-42 were both purchased from American Peptide Sunnyvale, CA, USA. AβpE3-42 was purchased from Bachem AG Bubendorf, Switzerland. 3NTyr10Aβ was provided to us by Clinical Neuroscience Unit, Department of Neurology, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany. 3NTyr10Aβ was made as described in [4]. We included this information in lines 133, 145 – 148 (unmarked copy)/lines 154, 168 – 170 (marked-up copy) of the revised manuscript. o Concentration of hexafluoroisopropanol in the Abeta 1-42 solution should be provided.For aliquotation Abeta 1-42 was dissolved in 100% hexafluoroisopropanol at a concentration of 1 mg/ml. As described above HFIP was completely removed after aliquotation and therefore no HFIP was present in the Abeta 1-42 solution that was injected. We once again apologize for the incorrect information about HFIP in the first version of the manuscript.o What was the oligomerization status of all 4 Abeta species at the time of injection? Was it comparable?All 4 Abeta species were injected 15 to 45 minutes after preparation. We assume that at this early timepoint after dissolving the substances the oligomerization process has just started and we injected predominantly Abeta monomers. Further oligomerization then should have taken place in the brains of the animals. A part of our study was to investigate whether this process leads to formation of Abeta plaques. As we were not able to detect Abeta plaques, retrospectively it would have been interesting to investigate for formation of Abeta oligomers e.g. using SDS-PAGE. We will consider this interesting approach when planning our next experiments.o Concentrations of antibodies used for WB should be provided.We apologize for not including this in the manuscript in the first place. The antibodies for WB were as follows: Primary antibodies Anti-Metabotropic Glutamate Receptor 5 Antibody ab53090 1:1000 Abcam TNFα Antibody ProSci XP-5284 1:1000 ProSci NMDAR2B Rabbit Antibody #4207 1:1000 Cell Signaling Technology Caspase-3 Rabbit Antibody #9662 1:1000 Cell Signaling Technology Secondary antibodies Anti-rabbit IgG, HRP-linked Antibody #7076 1:10 000 Cell Signaling Technology We corrected for this in lines 227, 228, 230 (unmarked copy)/lines 249, 250, 252 (marked-up copy) of the revised manuscript. o Why were the hippocampus and sensory cortex chosen for WB analysis?We wanted to investigate cognitive and behavioural changes in the modified hole-board test in wild type mice after intracerebroventricular injection of different Abeta subspecies as the primary endpoint of our study. As the hippocampus is important for working and reference memory and the sensory cortex plays a pivotal role in spatial orientation and movement planning, we decided to analyze TNF alpha, caspase 3, NR2B and mGlu5 as secondary endpoints in those brain regions.o How many saggital slices were analysed per mouse? Which brain regions were analysed?We analysed a total of 21 sagittal slices per mouse. The frontal and temporal lobe including sensory cortex and hippocampus were analysed. We included the number of brain slices and the location in line 198 (unmarked copy)/line 220 (marked-up copy) of the revised manuscript. Results and Interpretation• The results are currently unclear and could be improved:o Are the significant differences in the time trial data for the Abeta-pyro condition only for the 7-8th day of testing? Is the statistically significant difference between Abeta-pyro vs PBS for the sham or isoflurane condition or both?Thank you very much for these important remarks. We analysed the data derived from the modified hole-board test using general linear models for the factors subspecies for injection, anaesthesia (isoflurane or sham) and the within-group factor time and their interaction terms. The results show differences that are present over the whole test period of 8 days and not only on the 7-8th day of testing. We agree with the reviewer, that the symbols marking significance in figures 1 und 2 could have been confusing for the reader, changed the figures accordingly and added the description “Effect over all days of testing” in the legend. Statistical analysis revealed a significant difference for the factor “subspecies for injection” Therefore, the difference between A-beta pyro and PBS is present for all animals. To make this clearer for the reader, we added mean difference and partial eta-squared for assessment of the effect size in the manuscript . o Are the data described in line 232 and 233  of the manuscript for the sham condition, the anaesthetic condition or both? Is the difference only present for the 7-8th day of testing?Regarding this comment please be referred to our answer on your comment above. The difference is present over all days of testing. We corrected figure 2 accordingly.• Abeta-pyro is by far the most concentrated Abeta species when injected, could this explain why the other Abeta species had no/limited effects? And explain the interaction of only Abeta-pyro and isoflurane in the behavioural testing data? Opposite effects of picomolar and micromolar Abeta1-42 and Abeta1-40 have previously been reported . There are also many published papers reporting protective effects of Abeta that should be included in the discussion related to the Abeta-pyro results .Although we cannot rule out that the different concentrations of the Abeta species, with Abeta-pyro being the most concentrated could be the reason for our results, we think that the distinctive properties of Abeta pyro might explain our findings and the interaction of only Abeta-pyro and isoflurane. Please be also referred to our answer to the following critique as well as the third Reviewer comment in the Methods section. In order to improve the discussion of the Abeta effects we included the referenced studies in lines 351 – 354 and 371 – 374 (unmarked version)/lines 374 - 377 and 395 – 398 (marked-up version) of the revised manuscript. • Is there a potential mechanism for the interaction between Abeta-pyro and isoflurane to explain the results?The fact that most of the statistically significant results in our study could be found regarding Abeta-pyro might be due to the rather short time between the intracerebroventricular injection and the behavioural testing. Abeta-pyro has been shown to accumulate in the brain at early stages of AD , with the hippocampus being one of the predominant regions. 12-week-old C57BL/6 mice showed an impairment in spatial working memory and delayed learning in Y-maze and Morris water maze tests after intracerebroventricular injection of aggregated Abeta-pyro within two weeks after injection [20]. Abeta-pyro and isoflurane are both hydrophobic agents . As aggregated Abeta-pyro forms membrane pores and thus seems to alter membrane permeability [23], we also see a potential mechanism for the interaction there. We included our thoughts on a potential mechanism of interaction in the discussion (lines 363 – 365 (unmarked version)/lines 387 – 389 (marked-up version) of the revised manuscript). • Abeta1-42 is injected containing hexafluoroisopropanol and NaOH, there is no control group that accounts for the hexafluoroisopropanol and NaOH, thus, the difference in the time spent on the exposed part of the arena in the Abeta1-42 experimental group  may be due to the impact of hexafluoroisopropanol, NaOH, Abeta1-42 or a combination of the three.Once again, we are deeply sorry for not providing sufficient detail in the Methods section and would like to refer to our answer to the second reviewer comment on the methods. The Abeta 1-42 stock solution was purged from hexafluoroisopropanol (HFIP). NaOH in a concentration of 20 mmol/l was diluted 1:100 with PBS and reacted to sodium carbonate and sodium bicarbonate, so we think that the results are due to the impact of Abeta 1-42 rather than HFIP or NaOH. • The lack of difference in the amyloid deposits and TNFalpha, caspase 3, NR2B and mGlu5 may be due to the study being underpowered or a non-optimal concentration of Abeta species being used. This should be discussed.We fully agree with Reviewer #1 and would also like to refer to our answer to the first and third Reviewer comment in the Methods section. The primary endpoint of the study was the cognitive and behavioural outcome. The explorative studies we did on a limited number of brains on amyloidopathy, neuroinflammation and receptor expression should be considered preliminary. We stated this in lines 32, 250, 317, 382, 411, 412 (unmarked version)/lines 34, 273, 341, 406, 436, 437 (marked-up version) of the revised manuscript. • What were the differences between the previous study Schmid et al., 2017 (Reference 8) and the current study?The anaesthetic used for canula insertion surgery is different, the concentration of Abeta 1-42 injected is much higher, the number of mice per experimental group is higher, this warrants discussion.In the previous study of Schmid et al., 2017 mice were intracerebroventricularly injected with Abeta 1-42 or PBS. Subsequently neurocognitive and behavioural parameters were evaluated using the modified hole-board test. Mice were anaesthetized with a combination of midazolam, medetomidine and fentanyl intraperitoneally. The main differences between the study under review and the previous one are the different subspecies of amyloid-beta and the type of anaesthesia: since we wanted to investigate the effects of different amyloid-beta subspecies on cognition and behaviour, mice were intracerebroventricularly injected with not only Abeta 1-42 or PBS but with Abeta 1-40, Abeta 1-42, Abeta nitro, Abeta pyro and PBS. Also, we aimed to investigate a possible interaction between isoflurane anaesthesia and the respective amyloid-beta subspecies. We decided to also use isoflurane anaesthesia for cannula implantation to avoid any interaction of other anaesthetics and opioids like midazolam, medetomidine and fentanyl with the Abeta subforms.We agree that the concentration of Abeta 1-42 used in our 2017 study was higher. However, the final concentration of Abeta 1-42 in the brain is comparable as the amount of Abeta 1-42 is identical: In our 2017 study we injected 3.5 µl of a solution containing 1 µmol/l Abeta 1-42 resulting in a total amount of Abeta 1-42 of 3.5 pmol [24]. In the current study we injected 5.0 µl of a solution containing 700 nmol/l Abeta 1-42 resulting in the same total amount of Abeta 1-42 of 3.5 pmol. We agree that it was not clear for the reader at first sight that we injected 5.0 µl of Abeta and added the exact volume in the methods section (line 144 (unmarked version)/line 166 (marked-up copy) of the revised manuscript).We apologize for the confusion caused regarding the experimental groups. In the study of Schmid et al, 2017, a total of 24 mice was divided in 4 groups. The first group was injected with Abeta 1-42 and started testing on day 2, the second group  started on day 4, the third group  started on day 8 after the intracerebroventricular injection. The fourth group (mice injected with PBS) started on day 4 after the injection. In the current study we used 60 mice divided in 10 groups. In each study we used 6 mice per group. To make this clearer for the reader we included the exact number of animals per group in the manuscript (lines 122 - 125 (unmarked copy)/lines 143 – 146 (marked-up copy)).Other• The manuscript should use inclusive language for people living with dementia . This should be revised throughout. Thank you for this important comment. We revised the wording in lines 50, 60, 62 (unmarked copy)/lines 53, 64, 67 (marked-up copy) of the manuscript. • Figure 1 – reduce the range of the y axis so that data and error bars can be seen clearly.We apologize and revised figure 1 accordingly. • Figure 1, 2 – marking of significance on graphs is unclear, which time point is this for? Please rectify.We revised figures 1 and 2. Please be referred to our answer on your first comment in results and interpretation. • Line 232 remove the word “as”. We removed the word “as” in line 274 (unmarked version)/line 297 (marked-up copy) of the revised manuscript. • Line 268 “…an inhibiting effect on behaviour” this should be reworded to be more specific.We apologize for not expressing this more precisely and reworded the sentence in lines 313, 314 (unmarked copy)/lines 336, 337 (marked-up copy) of the revised manuscript. • Paragraphs 2 and 3 of the Discussion could be integrated for a better justification of the mouse model used.We integrated paragraphs 2 and 3 of the Discussion in lines 69 – 75 (unmarked version)/lines 70 – 85 (marked-up version) of the revised manuscript. • “in vitro” and “in vivo” should be italicised throughout. We italicised “in vitro” as well as “in vivo” in lines 80, 81, 153, 332, 347, 362, 363 (unmarked version)/in lines 94, 95, 176, 355, 370, 386, 387 (marked-up version) of the revised manuscript. Reviewer #2: The present study investigated the effect of different amyloid beta subspecies on behaviour and cognition in mice and their interaction with isoflurane anesthesia. The main findings of the study are that Aβ pyro improved overall cognitive performance which seemed to be contrary to the previous studies, and isoflurane could counteract this improvement. Inflammation and apoptosis biomarkers such as tumor -necrosis factor alpha, NR2B, mGlu5, or caspase 3 were not involved in this process. There are several points which the authors should consider.1. The biochemical endpoints appear to be limited to a single evaluation This would be informative to have additional time points to better understand the effects on these markers.We absolutely agree with Reviewer #2 that it would have been very interesting to perform biochemical analyses at several timepoints throughout our experiments. For example, given the fact that the caspase activation may not last very long following isoflurane anesthesia [7], it would have been informative to test for caspase 3 activity early after isoflurane anesthesia. However, the primary endpoint of our study was cognitive performance and behavioural alterations in the modified hole-board test. Like any other test in animals, the modified hole-board test is also very vulnerable regarding any interference. Exposing the animals to additional stressful situations, i.e. taking multiple blood samples, during the eight consecutive days of the modified hole-board testing would have altered the test performance of the mice. Therefore, we decided to evaluate the biochemical endpoints one time at the end of the mHBT being aware of this limitation to the study. To state this more clearly for the reader we reworded the corresponding paragraph in the discussion and emphasized the limitation of a missing consecutive analysis of biochemical parameters (lines 396 – 399 (unmarked version of the revised manuscript)/ lines 421 – 424 (marked-up copy of the revised manuscript)).2. Isoflurane is rarely used clinically, as it is known for its adverse effects on cognitive functions. It would be more pellucid if the authors could illuminate the reason why selected isoflurane in this study.Thank you for this important comment. We are sorry for not explaining this more clearly beforehand. As Reviewer #2 correctly stated, we wanted to investigate the effects of different amyloid beta subspecies on behaviour and cognition after intracerebroventricular injection in male C57BL/6N mice and their interaction with anaesthesia. We are aware, that isoflurane has adverse effects on cognitive function and is not regularly used in first world countries anymore. However, isoflurane is one of the most extensively studied anaesthetic agents in animal research. It has been shown to lead to increased oligomerization of amyloid beta in vitro  but not in vivo [27]. Since the above cited studies used human cell lines transfected with APP or a transgenic mouse model of Alzheimer’s disease, we decided to investigate the direct interaction of isoflurane and “extrinsic” amyloid beta in a mouse model of intracerebroventricular injection. We included our considerations regarding the use of isoflurane in the introduction of the revised manuscript (lines 78 - 82 (unmarked version)/ lines 92 – 96 (marked-up version)).We agree with the reviewer, that it would be very useful to examine different anaesthetics in future studies. Especially desflurane could be an interesting anaesthetic as it did not increase amyloid beta and tau levels in human cerebrospinal fluid and therefore could be regarded as less “neurotoxic” than isoflurane [9]. We included this consideration for future research in the manuscript (lines 404 – 406 (unmarked copy)/lines 429 – 431 (marked-up copy)).3. The size of the experimental groups of mice in the study is unclear. Is the number of animals included based on power analysis? Please state the number of animals in each experimental group in Material and MethodsWe are deeply sorry for not stating this more clearly. We included the number of animals in each experimental group  in Materials and Methods lines 122 – 125 (unmarked version)/lines 143 – 146 (marked-up version). Concerning a power analysis, please also be referred to Reviewer#1’s first comment in the Methods section. We apologize for not describing these very important considerations in the first version of our manuscript. In short, this study was designed as an observational study, with cognitive and behavioural outcome as primary endpoints. Therefore, sample size calculations were performed based on the following considerations: The variables of the hole-board test are considered relevant if two groups differ two times the given standard deviation. Based on a type I error of 0.05, a type II error of 0.20 and two-sided t-tests at the final test level of the hierarchical model 4 animals per group would have been appropriate. Our internal standard, however, suggests a minimal group size of six, which has been used accordingly. We added this information in lines 245 – 249 (unmarked version) of the revised manuscript/lines 268 - 272 (marked-up version) of the revised manuscript.References1. Mori H, Takio K, Ogawara M, Selkoe DJ. Mass spectrometry of purified amyloid beta protein in Alzheimer's disease. The Journal of biological chemistry. 1992;267(24):17082-6. Epub 1992/08/25.2. Saido TC, Iwatsubo T, Mann DM, Shimada H, Ihara Y, Kawashima S. Dominant and differential deposition of distinct beta-amyloid peptide species, A beta N3(pE), in senile plaques. Neuron. 1995;14(2):457-66. Epub 1995/02/01.3. Guix FX, Uribesalgo I, Coma M, Muñoz FJ. The physiology and pathophysiology of nitric oxide in the brain. Progress in neurobiology. 2005;76(2):126-52. Epub 2005/08/24.4. Kummer MP, Hermes M, Delekarte A, Hammerschmidt T, Kumar S, Terwel D, et al. Nitration of tyrosine 10 critically enhances amyloid β aggregation and plaque formation. Neuron. 2011;71(5):833-44. Epub 2011/09/10.5. 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Inhaled anesthetic enhancement of amyloid-beta oligomerization and cytotoxicity. Anesthesiology. 2004;101(3):703-9. Epub 2004/08/27.27. Bianchi SL, Tran T, Liu C, Lin S, Li Y, Keller JM, et al. Brain and behavior changes in 12-month-old Tg2576 and nontransgenic mice exposed to anesthetics. Neurobiology of aging. 2008;29(7):1002-10.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 13 Nov 2020Neurotoxicity of different amyloid beta subspecies in mice and their interaction with isoflurane anaesthesiaPONE-D-20-10009R1Dear Dr. Schmid,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Stephen D. Ginsberg, Ph.D.Section EditorPLOS ONE 25 Nov 2020PONE-D-20-10009R1 Neurotoxicity of different amyloid beta subspecies in mice and their interaction with isoflurane anaesthesia Dear Dr. Schmid:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. 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Several previous studies have reported a cross-sectional association between elevated high sensitivity C-reactive protein (hs-CRP) and migraine. The aim of this population-based follow-up study was to investigate the influence of hs-CRP at baseline on the risk of developing migraine 11 years later.Data from the Nord-Trøndelag Health Study performed in 2006–2008 (baseline) and 2017–2019 were used. A total of 19,574 participants without migraine at baseline were divided into three groups based on hs-CRP levels . Poisson regression was used to evaluate the associations between hs-CRP levels and risk ratios (RRs) of migraine, and precision of the estimates was assessed by 95% confidence interval (CIs).) was found, whereas no evident relationship was found between high hs-CRP level and risk of developing episodic migraine.In the multi-adjusted model, increased risk of migraine  was found in the highest hs-CRP levels group compared to the lowest group. In the group with the highest hs-CRP levels, a nearly three times higher risk of chronic migraine (RR 2.81, 95% CI 1.12–7.06The main finding in this 11-year follow-up was that hs-CRP levels between 10.00–20.00 mg/L at baseline was associated with increased risk of chronic migraine. The pathophysiology of migraine is complex and not fully understood . In the A review published in 2014 of previous cross-sectional studies evaluating the association between hs-CRP and migraine concluded that most studies have found increased hs-CRP levels , 4. HoweIn the present study, we evaluated the influence of hs-CRP on the risk of developing migraine in a 11-year follow-up. Based on previous knowledge, we hypothesized that elevated hs-CRP increased the risk of migraine..This is a population-based historical cohort study. The influence of hs-CRP at baseline was evaluated on the risk of migraine 11 years laterThe present study included data from the two last HUNT surveys conducted in Nord-Trøndelag County, Norway, in 2006–2008 (HUNT3) , and in n = 527)  Fig. .Fig. 1THUNT3 and HUNT4 had 14 identical headache questions . The inin = 293) and HUNT4 (n = 232) gave the following results: for migraine, the sensitivity was 54% and specificity 95%  and for migraine with aura, the sensitivity was 39% and specificity 95%  [We have previously reported the validity of the questionnaire-based headache diagnoses in HUNT3 , and HUN30–0.38) . For chrIn HUNT3, 50,803 persons (54%) participated out of 93,860 invited Fig. . Among t2); smoking ; alcohol consumption during the last year ; total Hospital Anxiety and Depression Scale , headache suffering (yes/no); self-reported diabetes (yes/no); self-reported stroke (yes/no); and self-reported hypertension (yes/no).The selection of potential confounders was based on previous literature –13. We eThis study was approved by the Regional Committee for Medical and Health Research Ethics, the Faculty of medicine, mailbox 8905, 7491 Trondheim. The approval number was #2018/2422/Rek Midt. The participants have given written informed consent.A modified Poisson regression with a robust error variance was used to estimate the association between hs-CRP at baseline and risk ratios (RRs) of migraine. Precision of RRs were assessed by 95% confidence intervals (CIs). We present results for three different statistical models separated by number of confounders included; model 1 (age and sex), model 2 , and model 3 . In model 3 we excluded factors if they did not change RR when evaluating each factor separately or when including several factors grouped together . Subjects with missing data on confounding factors  with hs-CRP levels < 3.00 mg/l were younger and more likely to have education ≥13 years, and less likely to report diseases or complaints than those with hs-CRP levels ≥3.00.Baseline characteristics of the population at risk related to hs-CRP categories in HUNT3 are given in Table n = 19,574), 766 (3.9%) fulfilled the criteria of migraine at follow-up in HUNT4, giving an incidence of 3.6 per 1000 person-years. A total of 354 (1.8%) had migraine with aura, 412 (2.1%) migraine without aura, 711 (3.6%) had episodic migraine) and 55 (0.3%) chronic migraine.In the population at risk , migraine with aura , and chronic migraine  (Table In this population-based 11-year follow-up study the main finding was an increased risk of chronic migraine in participants with high hs-CRP levels (10.00–20.00 mg/l) at baseline.Most previous migraine studies focusing on hs-CRP have been clinical-based with relatively low number of participants . For exaDuring the 11-year follow-up, the incidence of migraine was 3.6 per 1000 person-year. In comparison, a much higher incidence of migraine (8.1 per 1000 person-years) was found in a 12-years’ follow-up in Denmark . The reaThe main result in the present 11-year follow-up was that high hs-CRP was associated with increased risk of chronic migraine. This is of relevance in the question of inflammation as an involved mechanism in the transitation from episodic to chronic migraine , 19.The major strengths of this study are the longitudinal population-based cohort design with many participants, a wide age range, and the use of validated diagnoses of headache , 11. In Several study limitations should also be considered. Firstly, we have no information about potential risk factors of migraine that may have appeared during the follow-up period before HUNT4. Secondly, generalization of the results to the entire population must be made with some caution, since only 57% in the population at risk in HUNT3 were included in the present study (21% of the invited population in HUNT3), and we cannot be certain that loss to follow-up was random. Thirdly, although the validity of headache ≥15 days/month was good, it should be highlighted that only one person had chronic migraine among participants in the validation studies [9, 10). Thus, the validity of the questionnaire-based diagnosis of chronic migraine could not be reported. Finally, very few (55 participants) of the population at risk developed chronic migraine in HUNT4, giving a wide confidence interval of the estimated risk ratio. On the other hand, it should be highlighted that consistent results have been shown in two large cross-sectional studies [In this population-based cohort study participants with high hs-CRP levels at baseline had an increased risk of chronic migraine.


Chronic kidney disease (CKD) accelerates the development of neointima formation at the anastomosis site of arteriovenous (AV) fistulas. Accumulation of certain uremic toxins has a deleterious effect on the cardiovascular system. The oral charcoal adsorbent, AST-120, reduces circulating and tissue uremic toxins, but its effect on neointima formation at an AV fistula is unknown. To understand the effect of CKD and AST-120 on neointima formation, we created AV fistulas  in mice with and without CKD. AST-120 was administered in chow before and after AV fistula creation. Administration of AST-120 significantly decreased serum indoxyl sulfate levels in CKD mice. CKD mice had a larger neointima area than non-CKD mice, and administration of AST-120 in CKD mice attenuated neointima formation. Both smooth muscle cell and fibrin components were increased in CKD mice, and AST-120 decreased both. RNA expression of MMP-2, MMP-9, TNFα, and TGFβ was increased in neointima tissue of CKD mice, and AST-120 administration neutralized the expression. Our results provided in vivo evidence to support the role of uremic toxin-binding therapy on the prevention of neointima formation. Peri-operative AST-120 administration deserves further investigation as a potential therapy to improve AV fistula patency. Arteriovenous (AV) fistula dysfunction, such as stenosis and thrombosis, is a major cause of morbidity and mortality among hemodialysis patients. Native AV fistulas are the best vascular access of choice owing to the low incidence of stenosis, thrombosis, or infection as compared to prosthetic grafts or central vein catheters. However, failure rate of AV fistula within two years of creation is as high as 50% . In the AV fistulas are usually created for end-stage renal disease (ESRD) patients for the maintenance of hemodialysis. Chronic kidney disease (CKD), especially ESRD, has been recognized as a strong risk factor for cardiovascular disease. In animal models, CKD accelerates the development of intimal hyperplasia at the venous outflow tract near the AV anastomosis ,4. In adAST-120 is an oral carbon adsorbent that can adsorb at least six negatively charged and 17 positively charged uremic toxins, including IS and PCS precursors, thereby reducing serum IS and PCS levels ,9,10,11;p = 0.0001) and 12 weeks  after 5/6 nephrectomy (p = 0.02) and 12 weeks   C. There  = 0.03) D.2 vs. 209,983 ± 9885 μm2, average increase 35%, p < 0.001) at four weeks after AV fistula creation. The increase in neointima area of CKD mice was attenuated  in CKD mice fed with AST-120 . The increase in the IEL circumscribed area was neutralized in the CKD mice administrated with AST-120  (p = 0.99). The patency of the AV fistula at four weeks after creation was the lowest for the CKD mice (62%) and the administration of AST-120 improved the patency rate of the CKD mice (83%), similar to that of the non-CKD mice (86%)  C. There ce (86%) D. Accord2 vs. 17,443 ± 1779 μm2, average increase 59%, p < 0.001). The increase in α-SMA staining area of CKD mice was reversed if the CKD mice were fed with AST-120 .Immunofluorescence statin with α-SMA, fibrinogen, and collagen antibodies was used to discriminate the change in specific compositions of neointima tissues: . The imm2 vs. 11,806 ± 1065 μm2, average increase 77%, p = 0.01). The increase in the fibrinogen-stained area of CKD mice was reversed if the CKD mice were fed with AST-120 .Given that the primary thrombus was mainly composed of fibrin (nearly 60%), fibrinogen antibodies were used to evaluate the thrombus components in the neointima tissues . In the 2 vs. 74,175 ± 4459 μm2, average increase 73%, p = 0.01). The increase in collagen-stained area of the CKD mice was reversed if the CKD mice were fed with AST-120 .The collagen content of the neointima tissue was evaluated by the Picro Sirius stain . The CKDp = 0.036). The upregulation of TNF-α mRNA expression was reversed if the CKD mice were fed with AST-120 . There was no significant difference in gene expression of IL-6 and MCP-1 among the non-CKD mice, CKD mice, and CKD mice fed with AST-120.The effect of CKD and AST-120 on gene expression in AV fistula was demonstrated on p = 0.038), and it was attenuated by AST-120 in the CKD mice fed with AST-120 . Only a trend toward increasing expression of MMP-2 in CKD mice was observed, and AST-120 also attenuated the upregulation of MMP-2 in the CKD mice.CKD mice had an upregulated expression of MMP-9 mRNA compared to the non-CKD mice  in CKD mice was also surpassed if it was fed with AST-120.There was no significant difference in the change of pro-thrombosis gene expressions, such as tissue factor and PAI-1, among the three groups of mice (data not shown).The present study makes a novel observation that AST-120 therapy attenuated the development of neointima after AV fistula creation. This benefit was not dependent on the change in body weight or renal function. Instead, reduction of neointima formation after AST-120 therapy was associated with a parallel decrease in plasma IS levels and mitigated pro-inflammatory cytokines and metalloproteinase in neointima tissues.Several animal studies have shown that CKD aggravates neointimal hyperplasia in AV fistulas ,4. The sNeointima formation is a complex process involving VSMC proliferation, migration, and synthesis of the extracellular matrix. There is also a close link between neointima formation and endothelium dysfunction. Although we did not explore the detailed cellular or molecular mechanisms, previous studies gave mechanical insights for the effect of AST-120 therapy. It has been demonstrated in vitro that some uremic toxins, such as IS and p-cresyl sulfate, inhibit endothelial cell proliferation and endothelial function ,18. MoreAmong the uremic toxins, IS the most extensively studied with regard to their negative impact on the cardiovascular systems. IS could activate the immune cells and lead to the activation of inflammatory cytokines, such as TNFα and IL-6. In animal studies, AST-120 could reduce IS-related activation of inflammatory cells and oxidative stress . Uremic Recently, retained uremic solutes were found to be responsible for enhanced thrombogenicity in CKD milieu in ex-vivo experiments ,27,28. PIn addition to inward remodeling, an unexpected finding on outward remodeling of the CKD mice was also of interest. The CKD mice had a more prominent outward remodeling than the non-CKD mice, as reflected by a larger IEL area at four weeks after creation. Administration of AST-120 inhibits the outward remodeling of CKD mice, suggesting a positive impact on outward remodeling by uremic toxins. Blood flow-induced MMP activation is a critical pathway for both the inward and outward remodeling. On the one hand, breakdown of extracellular matrix and IEL fragmentation lead to the reconstruction of the vascular scaffold, facilitating the expansion of vessels. On the other hand, breakdown of extracellular matrix also helps the migration of smooth muscle cells into the subendothelial space. The matrix fragments released by MMP-9 also activate VEGF and TGF-β production and then increase the collagen production ,32. In oSeveral studies have investigated the effectiveness of pharmacological therapy to improve patency of AV fistula during the peri-operative period, including antiplatelets, anticoagulants, statins, fish oil, and anti-hypertensive agents. Nonetheless, systemic reviews or meta-analysis showed controversial or no beneficial effects ,35. CurrSome limitations should be addressed. Our study found that AST-120 treatment decreased circulating IS levels and lessened neointima formation. Nonetheless, the changes in IS level and neointima formation are not necessarily causally related. AST-120 is a non-specific adsorbent and a variety of uremic toxins could be removed, not only IS. Some of these toxins, such as PCS and advanced glycated end products, also have deleterious effect on vascular homeostasis. It was supported by our finding that the reduction of neointima volume and messenger RNA expression surpassed that of IS levels. Therefore, the precise mechanisms and molecular targets of AST-120 treatment still need to be clarified in future studies.Our study provides in vivo evidence supporting the role of uremic toxins on stenosis and thrombosis of AV fistulas. Furthermore, the positive results in the animal study warrant further exploration of the attenuation of uremic toxin-binding therapies on neointima development after AV fistula creation.All experimental procedures and protocols were pre-approved by the institutional animal care committee of Taipei Veterans General Hospital , approval code: IACUC 2014-108, date of approval: 2014/08/01. Six- to 8-week-old C57BL6/J male mice were purchased from the National Laboratory Animal Center . These mice were housed at 22 °C temperature, 41% relative humidity, and 12 h light-dark cycles, with access to food and water ad libitum prior to the experiments.Under intraperitoneal anesthesia with 2,2,2-tribromoethanol  at 200 mg/kg, both the upper and lower poles of the left kidney were removed through a lateral incision, and the cut-ends of the left kidney were cauterized to prevent bleeding. One week after the first operation, the right kidney was removed after ligation of the renal vessels and ureter under anesthesia . The micThe mice were divided into 3 groups: non-CKD group (shame operation), CKD group , and CKD+AST-120 group . One week after operation, 5% AST-120  were mixed in pulverized chow and fed to the mice of the CKD+AST-120 group to the end of the study at 12 weeks. The mice of the non-CKD and CKD group were fed with normal pulverized chow.Eight weeks after CKD creation, venous-end to arterial-side AV fistula was created according to the studies by Wong and Kang et al. ,43. The After AVF creation, the tissues samples were harvested on day 14 for gene expression analysis and on day 28 for histology analysis, according to the temporal change of intimal hyperplasia described by Wong et al. . The AVF4d (10 μg/mL) was used as an internal control and IS was detected by tandem mass spectrometry . The samples were sequentially injected into the UHPLC via the Acella 1250 autosampler (Thermo Fisher Scientific Inc.) and were separated using a Shiseido HPLC CAPCELL PAK C18 MGII column . The mobile phases were composed of (A) 0.1% (v/v) FA in water and (B) 0.1% (v/v) FA in MeCN, with a 250 μL/ min flow rate. The MS detection mode was set with an applied voltage of 2.5 kV in the negative ion mode, and the vaporizing and capillary temperatures were set at 300 °C and 350 °C, respectively. The survey scan mode “multiple reaction monitoring (MRM)” was utilized, with MRM transitions 212 > 80 and 212 > 132 belonging to IS and 216 > 80 and 216 > 136 belonging to IS-4d for quantification. The Xcalibur software  was used to acquire the MS spectra and control the mass spectrometer.The concentration of blood urea nitrogen and serum creatinine were determined by an autoanalyzer . Supernatant in mice serum samples (50 μL) was lyophilized, and then reconstituted in 400 μL 30% acetonitrile (ACN) aqueous solution with 0.1% formic acid (FA) after protein precipitation. IS-2). The AVFs were defined as patent histologically when the maximum stenosis percentage in all included venous sections was ≤95%.The tissue was stained with Weigert’s elastin stain . The venous segment at 600 μm downstream to the anastomosis was sectioned at 5 μm thickness, and the most severe sections were used for the analysis . After d2) for specific components. For immunohistochemical staining, the primary antibody against TNF-α and MMP-9  was used. The sections were visualized with 3,3′-diaminobenzidine tetrahydrochloride  and counterstained with hematoxylin.Paraffin-embedded tissues were stained with H&E, and then morphometric analysis was performed. Sections were stained using primary antibodies. For the immunofluorescence stain, primary antibodies against the α-smooth muscle actin  and fibrinogen  were used to delineate the smooth muscle cell and thrombus components in the neointima area. The morphometric analysis was performed using the Image J software and expressed as positive area , and the quality of RNA was confirmed by the 260–280 nm ratio. The SuperScript III First-Strand Synthesis Supermix  was used for reverse transcription. SYBR Green Supermix  was used for real time quantitative PCR, with a 35 cycles amplification using the iQ5 Real Time PCR Detection System. Primer efficiencies were determined by the melt curve analysis. All samples were normalized by amplification of a housekeeping gene RNA. The sequences of primers used in this study are provided in the Statistical analysis was performed using GraphPad Prism (version 7) software. All data are expressed as means ± SEM. One-way ANOVA with post-hoc analysis was performed for morphometric, immunofluorescence, immunohistochemical, and Collagen staining in the qPCR experiment. The primary endpoint was the difference of neointima volume. According to previous studies of CKD mice, 6 mice in each group were estimated to achieve a statistical power of 80% to detect a 50% difference at an α level of 0.05 . P value